Genetic analysis of the RcsC sensor kinase from Escherichia coli K-12

Abstract

The Rcs two-component pathway is involved in the regulation of capsule production in Escherichia coli. RcsC is predicted to be the sensor component of this two-component pathway, and in this study we present the first genetic data that support the role of RcsC as a hybrid sensor kinase.

FIG. 1.

Induction of cpsB-lacZ expression by DjlA overproduction requires an intact copy of rcsC. PSG1031 (rcsC⁺ cpsB-lacZ) and PSG1038 (rcsC52::Tn10 cpsB-lacZ) were transformed with pPSG961-31 (paraBAD-djlA) and, where indicated, pPSG980 (p rcsC⁺). Cells were cultured overnight at 30°C on LB agar plates in the presence of 0.2% (wt/vol) l-arabinose. To determine the level of cpsB-lacZ expression, cells were harvested and assayed for β-galactosidase activity. The results shown are the means of five independent measurements, and the error bars represent standard deviations.

FIG. 2.

yojN rcsCB genetic locus. (A) Arrangement of genes in the yojN rcsCB locus in E. coli K-12. Arrows, approximate positions of promoters implicated in the expression of each gene. (B) The modular architecture of RcsC. The input domain spans the cytoplasmic membrane and contains two transmembrane domains (TMD). The numbers flanking the transmitter domain and receiver domain represent the predicted amino acid sequence limits of each signaling domain based on in silico homology searches.

FIG. 3.

Identification of the amino acids predicted to be involved in the RcsC phosphorelay. (A) Alignment of the histidine-containing region (i.e., the H box) of the transmitter domain (H1) from E. coli sensor proteins EnvZ, TorS, and RcsC. Asterisk, position of the His residue known to be phosphorylated in EnvZ and TorS, which aligns with H463 in RcsC. (B) Alignment of the receiver domain (D1) of RcsC with the receiver domains of CpxR, OmpR, RcsB, and TorR. Asterisk, Asp residue that has been shown, either genetically or biochemically, to be phosphorylated in CpxR, OmpR, RcsB, and TorR. This residue clearly aligns with D859 in RcsC. Both alignments were carried out using the ClustalW algorithm at the European Bioinformatics Institute (http://www.ebi.ac.uk).

FIG. 4.

RcsCH463 and RcsCD859 are required for the induction of cpsB-lacZ expression associated with DjlA overproduction. (A) PSG1038 (rcsC52::Tn10 cpsB-lacZ)/pPSG961-31 (paraBAD-djlA⁺) was transformed with either pTRC99a (vector) or derivatives carrying different mutant alleles of rcsC,rcsC⁺ (pPSG980), rcsCH463Q (pPSG980H463Q), or rcsCD859Q (pPSG980D859Q). Cells were cultured for 20 h at 30°C on LB agar supplemented with 0.2% l-arabinose and assayed for β-galactosidase activity. The results shown are the means of four independent measurements, and the error bars represent standard deviations. In some cases the error bars are too small to be shown on the graph. (B) PSG1038 (rcsC52::Tn10 cpsB-lacZ) cells carrying plasmids expressing different c-myc-tagged alleles of rcsC were grown as outlined in the text and collected by centrifugation, and 0.2 A₆₀₀ equivalents were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting with a monoclonal antibody that recognizes the c-myc epitope. Lane 1, pBMM101 (vector); lane 2, pBMM102 (rcsC-myc); Lane 3, pBMM103 (rcsCH463Q-myc); Lane 4, pBMM105 (rcsCD859Q-myc). The arrow indicates the RcsC band that cross-reacts with the anti-myc monoclonal antibody.

FIG. 5.

RcsCD859 is required for complementation of the rcsC137 allele. SG20907 (rcsC137 cpsB-lacZ) was transformed with either pTRC99a (vector) or derivatives carrying different rcsC alleles, as indicated. Cells were grown on LB agar at 30°C for 20 h and assayed for β-galactosidase activity. The results shown are the means of two independent measurements, and the error bars represent standard deviations. In some cases the error bars are too small to be shown on the graph.

FIG. 6.

RcsCH463Q and RcsC-D1 interfere with normal signaling from the RcsC protein. PSG1031 (rcsC⁺ cpsB-lacZ)/pPSG961-31 (paraBAD-djlA) was transformed with either pTRC99a (vector) or derivatives carrying different mutant alleles of rcsC, as indicated. Cells were cultured for 20 h at 30°C on LB agar supplemented with 0.2% l-arabinose and assayed for β-galactosidase. The results shown are the means of four independent measurements, and the error bars represent standard deviations. In some cases the error bars are too small to be shown on the graph.

FIG.7.

In vivo intermolecular complementation between rcsCH463Q and rcsCD859Q. PSG1038 (rcsC52::Tn10 cpsB-lacZ)/pPSG957 (djlA⁺) was transformed with either pPSG980H463Q (rcsCH463Q), pBMM110 (rcsCD859Q), or both plasmids together (rcsCH463Q rcsCD859Q). Cells were cultured for 20 h at 30°C on LB agar and assayed for β-galactosidase activity. The results shown are the means of four independent measurements, and the error bars represent standard deviations.